A human replication initiation protein, Cdt1, is a central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by direct binding. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example, by geminin silencing with small interfering RNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. We established a high throughput screening system based on a modified enzyme linked immunosorbent assay to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we screened inhibitors from natural materials containing food components, and found that a glycolipid, sulfoquinovosyl diacylglycerol (SQDG), from spinach can inhibit Cdt1-geminin interaction in vitro, with 50% inhibition observed at concentrations of 1.79mug/ml. Other major glycolipids, such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) from spinach, had no influence. Surface plasmon resonance analysis demonstrated that SQDG bound selectively to Cdt1, but did not interact with geminin. Using three-dimensional computer modeling analysis, SQDG was considered to interact with the geminin interaction interface on Cdt1, and the sulfate group of SQDG was assumed to make hydrogen bonds with the residue of Arg346 of Cdt1. These data could help to further understanding of the structure and function of Cdt1. In addition, SQDG could be a clue to developing more appropriate inhibitors of Cdt1-geminin interactions.